This revision of an application for a SC2 Pilot Project Award is from young investigator who proposes to develop a non-invasive method for imaging neurons in tissue at sufficient resolution to study myelin structure. If successful, this research could potentially have a major impact on the study of neuro-regeneration and neuro-degenerative diseases. The reviewers really liked this well-written application. Its strengths include the novelty of the imaging method, the project's significance and potential impact, the outstanding qualifications of the PI and his mentors, and the strong development and mentoring plans. Additional strengths were the innovative approach, the well thought out aims, and the already developed imaging system for the studies. A few minor weaknesses were also noted. These included inadequate discussion of relevant developments in the imaging field, of the likelihood that sufficient resolution can be attained, and of the limits of light transmission in tissues. It was felt that te PI had responded well to most of the criticisms of the previous reviewers. Overall, the review panel felt that this is an excellent application from a well-trained young investigator. Although the project is somewhat risky, the potential impact of the results is significant. The proposed research is highly suited to the SC2 mechanism, and the excellent mentoring plan suggests that the PI will significantly benefit from an SC2 Award. ABSTRACT: Myelin is a specialized membrane around axon, which does not only play a significant role in neuronal signaling but is also implicated in several clinical conditions, such s multiple sclerosis and Charcot-Marie- Tooth disease. The complex interaction between axon and glial cells is the crucial mechanism that maintains an optimal level of the myelin thickness with respect to the axon diameter, or g-ratio. However, the dynamic process of myelin formation is not well understood partly because of the lack of technology for measuring the myelin thickness in vivo. Here we propose to develop a non-invasive, imaging-based methodology to determine the thickness of myelin and the g-ratio without exogenous labeling. Our method relies on a nonlinear optical signal generated around the lamellae of myelin. Unlike previous methods, our novel technique will provide micrometer-resolution structural information in live cells and tissue, therefore suited for longitudinal studies of the dynamics of myelination, demyelination, and remyelination. We will undertake appropriate initial experiments in order to validate the correlation between the new method and the myelin structure. First we will test the sensitivity of our imaging using a myelinating co-culture system. Secondly, we will study the polarization-sensitivity as a route to identify the myelin domains. Finally, we will investigate the correlation between our metric and g-ratio using a developing animal model. We anticipate that our proposed research will facilitate in vivo studies of the dynamic interaction between glial cells an axon with minimum animal sacrifice. Moreover, our development may also have a clinical relevance as a diagnostic for demyelination diseases. PUBLIC HEALTH RELEVANCE: Relevance Statement The health of myelin in the nervous systems is implicated in a variety of clinical conditions, such as multiple sclerosis and Charcot-Marie-Tooth disease. Currently there is no method to investigate the dynamics of the myelin biophysics in live animals with micro-meter resolutions, hampering further understanding of the diseases. The objective of this research is to develop a novel imaging-based method capable of non-invasive determination of the thickness, or g-ratio, of myelin in vivo and to develop algorithms to improve the precision.